Inflammatory Mediators and Modulators of Pain

G Protein–Coupled Receptors

Many mediators produced during inflammation, such as bradykinin, serotonin, prostaglandins, and chemokines, act via GPCRs. These receptors elicit a specific biochemical response that depends on the type of G protein that is activated. Activation of G _s stimulates adenylate cyclase to raise the level of cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA) in the neuron, whereas G _i inhibits the activity of adenylate cyclase to lower cAMP levels. Although many cAMP effects are mediated by PKA, other mechanisms may be operative. For example, cAMP can activate Epac (exchange protein directly activated by cAMP), a guanine nucleotide exchange factor, which leads to activation of the ϵ isoform of protein kinase C (PKC-ϵ). Stimulation of G _q/11 activates phospholipases, notably phospholipase C (PLC), which generates inositol triphosphate (IP ₃ ) and diacylglycerol (DAG) from the membrane lipid precursor phosphatidylinositol 4,5-bisphosphate (PIP ₂ ). G _q activation can also stimulate PLA ₂ , which cleaves membrane phospholipids at the sn-2 position to produce the prostaglandin precursor arachidonic acid. G-protein control of cellular function can also involve direct action of βγ subunits on ion channels and enzymes, such as PLC (see ).

Ion Channels

Some inflammatory mediators act by directly gating the ion channels expressed by sensory neurons. Notable examples in this class are adenosine triphosphate (ATP; acting via P2X channels), protons (acting via acid-sensing ion channels [ASICs] and transient receptor potential vanilloid 1 [TRPV1]), and the lipid activators of TRPV1. All these ion channels are cation selective and are permeable to either sodium ions or both monovalent and divalent cations. In all cases the ion flow evoked by channel opening depolarizes the sensory neurons and leads to neuronal firing.

Receptor Tyrosine Kinases

The third general type of receptor includes cytokine receptors activated by mediators such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α) and the receptor tyrosine kinases for neurotrophic factors, such as the receptors for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and artemin. Both classes of receptors have monomers derived from a single transmembrane segment with a large extracellular ligand-binding domain. The cytosolic domain of receptor tyrosine kinases contains an intrinsic protein tyrosine kinase catalytic site, whereas the cytosolic domain of cytokine receptors is generally associated with a separate protein kinase that is recruited to the complex either directly or via adapter proteins. The functional receptors are either dimers or trimers, which either exist normally or are formed by cross-linking of adjacent monomers by the ligand. In either case, ligand binding activates kinase pathways that affect gene transcription and can also elicit acute effects on neuronal function.

Nitric Oxide and Cyclic Guanosine Monophosphate

In addition to receptor-mediated signaling, cells also signal via nitric oxide (NO). NO is an important intercellular mediator and is produced by many cells that have close physical association with neurons both in the periphery and within the spinal cord. NO is formed from l -arginine following activation of the enzyme nitric oxide synthase (NOS) by calcium and other co-factors, including calmodulin. Three forms of NOS have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), each with a distinct physiological role. nNOS and eNOS are both Ca ²⁺ /calmodulin dependent and are present in both the spinal cord and brain, whereas iNOS is functionally Ca ²⁺ independent and normally expressed in macrophages, inflammatory cells, and glia (for review see ). NO diffuses to its site of action, where it stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP). In turn, cGMP modifies intracellular processes, including activation of protein kinases, ion channels, and phosphodiesterases. NO can also act in other ways, for example, by activating cyclooxygenase (COX) enzymes and by S -nitrosylation of proteins ( ).

Bradykinin Receptor Signaling

B ₁ and B ₂ receptors couple through G _q α to stimulate PLC, which results in phosphoinositide hydrolysis, DAG production, and mobilization of intracellular Ca ²⁺ from intracellular stores. They can also act through G _i α to inhibit adenylate cyclase and stimulate the MAPK pathways ( ). A significant body of evidence supports the idea that bradykinin activates sensory neurons via a DAG–PKC pathway. Bradykinin causes the translocation of a specific PKC isoform, PKC-ϵ, from the cytoplasm to the plasma membrane of dorsal root ganglion (DRG) neurons ( ), and the excitatory effects of bradykinin are inhibited by the PKC inhibitor staurosporine ( ), which also attenuates the responses of skin afferents ( ). Furthermore, the bradykinin responses of many, but not all, neurons are reduced or abolished when PKC activity is down-regulated by prolonged exposure to phorbol esters ( ).

PKC activators depolarize sensory neurons by opening a cation-permeable ion channel ( ), and several pieces of information indicate that bradykinin exerts its effects, in part, by sensitizing or opening the heat-sensitive TRPV1 ion channel. Bradykinin activates ion channels in DRG neurons with properties similar to those of TRPV1 channels ( ); this agonistic effect requires the presence of PKC-ϵ and is blocked by PKC inhibitors ( ). Bradykinin also increases the capsaicin sensitivity of TRPV1 and reduces the temperature threshold for activation from approximately 42°C toward or below normal body temperature via a PKC mechanism ( ).

Activation of TRPV1 cannot explain all the excitatory effects of bradykinin inasmuch as activation of vagal and visceral afferents by bradykinin is retained in TRPV1 knockout mice ( ) and bradykinin can stimulate DRG neurons from TRPV1 ^−/− mice ( ). Bradykinin can also act via PLC to activate TRPA1 ( ), and bradykinin-evoked responses were significantly attenuated in sensory neurons from both TRPV1 and TRPA1 knockout mice ( ). One possibility is that TRPV1 and TRPA1 act in concert. In this scenario ( ), activation of PLC evokes TRPV1 gating and calcium influx. Because TRPA1 is often co-expressed with TRPV1 and because TRPA1 can be activated by increases in the intracellular calcium concentration ( , Zuborg et al 2007), a small calcium influx through TRPV1 may activate TRPA1.

Failure to inhibit bradykinin responses in all sensory neurons with staurosporine or prolonged exposure to phorbol esters ( ) suggests that excitation can be mediated by a PKC-independent mechanism. Other evidence points to different phospholipase-linked mechanisms resulting in activation of TRPV1. One proposal is that binding of PIP ₂ to TRPV1 inhibits channel activity ( ) and its hydrolysis by B ₂ receptor–mediated activation of PLC potentiates channel opening by removing this tonic inhibition ( ). However, the inhibitory influence of PIP ₂ on TRPV1 has been challenged, and there is evidence that PIP ₂ binding potentiates rather than inhibits TRPV1 ( ). Phosphoinositide binding may have both inhibitory and potentiating effects on TRPV1, depending on the level of stimulation ( ).

B ₂ receptor activation also stimulates the 12-lipoxygenase pathway and leads to the production of endogenous TRPV1 agonists (e.g., 12-hydroperoxyarachidonate [HPETE] and leukotriene B ₄ [LTB ₄ ]. Bradykinin-evoked activation of TRPV1-like currents, neuronal firing, and behavioral responses are blocked by lipoxygenase inhibitors, consistent with a contribution of this pathway ( ). Other data point to a role of COX products since the COX inhibitor flurbiprofen inhibits the heat sensitization induced by bradykinin in a skin–nerve preparation ( ).

Two other ionic mechanisms have recently been proposed for bradykinin-evoked activation of DRG neurons. Depolarization resulting from inhibition of M-type potassium currents and activation of a calcium-activated chloride current, encoded by TMEM16A, have been proposed as important PLC-linked mechanisms for the excitatory actions of bradykinin ( ).

Prostaglandins

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX enzymes, are the most widely used and effective drugs for the clinical treatment of inflammatory pain and hyperalgesia. NSAIDs have no obvious effect on normal pain thresholds but attenuate the abnormal pain responses in inflammatory conditions. Two COX enzymes, COX-1 and COX-2, are responsible for the first steps in prostaglandin synthesis. These enzymes have two catalytic enzymatic activities: a COX activity responsible for the production of PGG ₂ from arachidonic acid and a peroxidase activity that reduces PGG ₂ to form PGH ₂ , the first steps in prostanoid biosynthesis.

In general, COX-1 is considered to have a “housekeeping” role in almost all tissues mediating physiological responses. In contrast, COX-2 is not constitutively expressed (except in the kidney, vas deferens, and importantly, the brain) but is induced in inflammatory conditions. In the periphery, COX-2 expression is induced in cells involved in inflammation (macrophages, monocytes, and synoviocytes) and is primarily responsible for synthesis of the prostaglandins involved in acute and chronic inflammatory states. COX-2 expression is induced in peripheral tissues in animal models of arthritis, and up-regulated expression is seen in human rheumatoid arthritic joints, although relatively little expression has been noted in human osteoarthritic joints. Both COX-1 and COX-2 are expressed constitutively in DRG neurons and in the spinal cord. Normally, COX-1 is expressed in small and medium-sized DRG neurons and in neurons and astrocytes in the spinal cord. Enzyme expression in both neuronal and non-neuronal cells in the spinal cord is up-regulated after peripheral inflammation and nerve injury (see ), and intraspinal release of PGE ₂ is enhanced during peripheral inflammation ( ).

The important roles of spinal cord COX enzymes are not discussed in detail here but are covered in Chapter 28 . The available information indicates that COX inhibition at both peripheral and central sites can contribute to the anti-hyperalgesic effects, with the predominant clinical effect being mediated centrally. Certainly, prostaglandins produced in the periphery after tissue injury can sensitize peripheral nerves and induce hyperalgesia in animal models of inflammation, thus suggesting that a component of hyperalgesia could be due to a peripheral action. However, the finding that intrathecal administration of COX-2–selective inhibitors suppresses experimentally induced inflammatory hyperalgesia also argues for a central site of action ( ). The observations that COX-2 inhibitors have clinical efficacy similar to that of non-selective NSAIDs and that COX-2 inhibitors exert a rapid effect after surgery also argue that they act in these conditions at central sites where COX-2 is constitutively expressed.

PGH ₂ is metabolized by different prostaglandin synthetases to a range of prostaglandins. Prostaglandins such as PGE ₂ , PGD ₂ , and PGI ₂ are produced during inflammation and act with some specificity on different prostanoid receptors, termed EP, DP, and IP, respectively. Each of the prostanoid receptors has distinct coupling to G proteins, and the pattern of coupling determines the biochemical consequence of receptor activation. Four major types of EP receptors (EP _1–4 ) have been described, and splice variants of the EP ₃ subclass have also been identified, which probably explains the multiplicity of transduction pathways that have been associated with this receptor. In situ hybridization studies have shown the presence of mRNA for IP, EP ₁ , EP ₃ , and EP ₄ receptors in DRG neurons. About half the neurons express EP ₃ receptor mRNA; 40%, IP mRNA; 30%, EP ₁ mRNA; and 20%, EP ₄ mRNA, with some degree of co-expression ( ). Of these, EP ₁ , EP ₄ , IP, and some splice variants of EP ₃ receptors (EP _3B and EP _3C ) couple positively via G _s to stimulate adenylate cyclase and raise cAMP levels.

A major peripheral effect of PGE ₂ and PGI ₂ is to sensitize afferent neurons to noxious chemical, thermal, and mechanical stimuli (see, for example, ). In contrast, PGD ₂ shows little or no such activity ( ). The importance of these receptor subtypes in the periphery is confirmed by the findings that EP ₃ ^−/− and IP ^−/− mice show reduced hyperalgesia after lipopolysaccharide (LPS) administration ( ). In contrast, intrathecal administration of PGE ₂ induced normal mechanical allodynia in wild-type and EP ₃ ^−/− mice but not in EP ₁ ^−/− mice, thus illustrating that the EP ₁ receptor plays a role in prostaglandin-induced spinal sensitization ( ).

Lipoxygenase Products

The potential role of lipoxygenase products in inflammatory pain is less clear, and although the levels are increased in inflammatory conditions, evidence of a direct role in nociception is lacking. The major effect of these lipids is to recruit immune cells and alter microvascular permeability. Intradermal injection of LTB ₄ or 8R,15S-diHETE decreases mechanical and thermal thresholds in rats ( ; ; ) and humans ( ), and LTB ₄ sensitizes dental afferents ( ). The sensitizing actions of LTB ₄ require the presence of polymorphonuclear (PMN) leukocytes and are thus likely to be indirect ( ). 8R,15S-diHETE reduces the thermal and mechanical thresholds of C fibers ( ) and excites some C-fiber neuromas ( ). A role of LTB ₄ in experimental antigen (ovalbumin)-induced mechanical hyperalgesia has been shown by using the LTB ₄ antagonist CP10596 ( ). More recently, the cysteinyl-leukotriene receptor CysLT2 was found to be expressed in about 40% of rat DRG neurons, preferentially in small-diameter neurons. Intraplantar administration of the CysLT2 agonist LTC ₄ strongly enhanced the nocifensive response evoked by the P2X ₃ agonist αβ-me-ATP but was without effect on thermal sensitivity, thus suggesting a lack of effect on TRPV1 channels ( ).

One probable action for some lipoxygenase products is to activate TRPV1 channels inasmuch as 12S-HPETE, 15S-HPETE, 5S-HETE, and LTB ₄ all open TRPV1 channels in DRG neurons ( ). The behavioral effects of 8R,15S-diHETE noted earlier are unlikely to be due to such an action since this lipid shows very weak agonist effects on TRPV1.

Other Fatty Acid Metabolites

In addition to the leukotrienes, lipoxygenases can also convert eicosapentaenoic and docosahexaenoic acids into active signaling molecules. Formation of some of these metabolites requires the sequential action of COX-2 or cytochrome P450 followed by lipoxygenase-mediated oxidation ( ). The resulting molecules have been named resolvins because of the roles that they are thought to play in the resolution phase of inflammation, and they have attracted interest for their analgesic potential ( ). The resolvins RvE1 and RvD1 potently reduce thermal and mechanical hypersensitivity in inflammatory pain models. Resolvins produce these effects by stimulating G _i/o -coupled GPCRs located both on DRG neurons and in the spinal cord, thereby effectively inhibiting the activity of the sensory neuron ion channels TRPA1 and TRPV1, as well as C-fiber evoked long-term potentiation in the spinal cord ( ).

Linoleic acid is converted into several hydroxyl and carbonyl derivatives (9-HODE, 13-HODE, 9-oxoODE, and 13-oxoODE) by both lipoxygenase pathways and non-enzymatic lipid peroxidation reactions. In experimental situations, formation of these mediators is increased by depolarization of the spinal cord with a high-K ⁺ solution ( ). Extended exposure to heat also significantly increases the tissue concentration of these oxidized linoleic acid metabolites in mouse skin biopsy samples. Application of 9-HODE to cultured trigeminal neurons stimulates TRPV1, and administration in vivo evokes nocifensive behavior and thermal hypersensitivity, which is absent in Trpv1 ^{− / −} mice, thereby demonstrating that TRPV1 mediates the nociceptive effect of 9-HODE (Patwardhan et al 2010). Thus, oxidized linoleic acid metabolites, such as the endocannabinoid anandamide and several lipoxygenase products formed from arachidonic acid, can act as direct TRPV1 agonists ( ).

During conditions characterized by oxidative stress, such as inflammation or reperfusion after ischemia, a range of lipid peroxidation products are formed in reactions between free radicals and membrane lipids. Many of the lipids formed are well-known reactive, electrophilic molecules that bind covalently to proteins such as hydroxynenonal, cyclopentenone prostaglandins, isoprostanes, and related species. The covalent modification of redox-sensitive transcription factors initiates specific signaling cascades that may act to modify or protect against oxidative conditions, but the electrophilic lipids also stimulate nociceptive sensory neurons directly by activating TRPA1 ( ).